The applicant will attempt to determine how exercise enhances and denervation inhibits the ability of insulin to stimulate glucose transport and glycogen synthesis in skeletal muscle. They propose two novel hypotheses. One hypothesis links alterations in the energy expenditure of the muscle cell such as occur during exercise or inactivity, to changes in the concentrations of malonyl CoA and cytosolic long chain fatty acyl CoA (LCFA Co A). They speculate that changes in the concentrations of these metabolites lead to alterations in the activity of one or more PKC isozymes and other events that affect insulin signaling, and its ability to stimulate glycogen synthesis. The second hypothesis focuses on a pool of GLUT-4 glucose transporters, possibly associated with muscle glycogen, that has recently been identified by Dr. Pilch. They propose that transporters from this pool are mobilized predominantly during exercise, but once mobilized can be recruited by insulin. If so, this pool could account for both the enhancement of insulin-stimulated glucose transport after exercise and the decrease in insulin-stimulated glucose transport in denervated and immobilized muscle. These hypotheses will be tested using intact rodents and perfused and incubated rodent muscles as models. The specific aims are: 1) To characterize the temporal interrelations between changes in malonyl CoA and other metabolites, the distribution and activity of PKC isozymes and insulin signaling and action in denervated (inactive) muscle. They will also study the distribution of GLUT-4 between the exercise-and insulin-recruitable pools in this model. 2) To determine whether prior exercise causes changes in signal transduction and GLUT-4 distribution opposite to those produced by denervation and limb immobilization. 3) To compare the mechanisms by which insulin-stimulated glucose transport and glycogen synthesis are inhibited in denervated muscle and muscle perfused or incubated for many hours with a hyperglycemic medium. These studies should provide insights into the mechanisms by which contractile activity and hyperglycemia alter the action of insulin on muscle. They should also result in the development of appropriate model systems for examining the effects of insulin and other agents on muscle metabolism and signal transduction, in vitro.